Aim of this paper was that to prepare biocompatible, polyaspartamide based copolymers containing spermine or spermine/hydrophobic side chains able to condense nucleic acids and to transfect mammalian cells. Copolymers were prepared starting from alpha,beta-poly-(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) and exploiting the reactive hydroxyl groups in the polymeric side chains by subsequent activation reactions to obtain PHEA-Spermine (PHEA-Spm) and PHEA-Spermine-Butyramide (PHEA-Spm-C(4)). Molecular, physico-chemical and biological characterization of copolymers and interpolyelectrolyte complexes with plasmid DNA was performed. Experimental results evidenced that these copolymers are able to form complexes with plasmid DNA already at low polycation/DNA weight ratio ranging from 0.75/1 to 2/1. Interpolyelectrolyte complexes with decreased size were obtained when increasing the polycation/DNA weight ratio, until nanosized dimensions were reached. Copolymers as well as complexes were not haemolytic and non toxic in vitro. In vitro cell transfection with PHEA derivatives showed good biocompatibility and high transfection efficiency (luciferase) in cancer cells in comparison with commercially available, but toxic transfection agents.